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We also recorded activity of 26 nearby ARC neurons that were significantly and strongly suppressed (by at least 20%) by photostimulation (Figure 1E and Figure 1��figure supplement 1B; see also ��Materials and methods��). Because the only EPZ5676 ARC neurons currently known to be inhibited by AgRP neurons are POMC neurons (Cowley et al., 2001, Atasoy et al., 2012), a large fraction of ARCinh neurons are likely to be POMC neurons. We also recorded from an additional 33 nearby neurons that were unaffected by photostimulation (��ARCother��). All recordings were performed in mice habituated to head restraint (see ��Materials and methods��), as this enabled recordings with greater stability from a larger number of electrodes. Opposing modulations in ARC neuron firing across times of day In a first experiment in ad libitum-fed mice, we measured the firing of ARC neurons during daily 1-hr recording sessions at different phases of the light period, as the stomach is emptying (Kentish et al., 2013), levels of ghrelin, a hormone known to increase AgRP neuron activity, are rising (Tschop et al., 2000, Cummings et al., 2001, Wang et al., 2002, Bodosi et al., 2004), and minimal feeding is occurring as compared to the subsequent dark period (Lu et al., 2002). Stable firing across tens of minutes (Figure 1F) allowed reliable estimation of mean firing rate. As predicted by diurnal variations in in vitro AgRP neuron activity (Yang et al., 2011, Krashes et al., 2013) and in ARC expression of Agrp mRNA (Lu et al., 2002), learn more AgRP neurons demonstrated a significant, approximately fivefold increase in firing in afternoon vs morning recordings (p = 0.001; n = 10 vs 9 neurons, respectively; Figure 2A). In contrast to AgRP neurons, we observed a trend towards decreased firing in afternoon vs morning recordings across all non-AgRP neurons (p = 0.09, n = 32 neurons), with ARCinh neurons showing a similar trend (p = 0.13, n = 14; Figure 2A). Figure 2. Arcuate neurons demonstrate changes in firing rate across the light period. AgRP neuron photostimulation of food-seeking and food intake has been shown to be dependent on stimulation frequency, with greater potency at 20 Hz than at 5 Hz Oxacillin or 10 Hz (Aponte et al., 2011). Further, the release of peptides is also likely to depend on spike frequency (Summerlee and Lincoln, 1981, Aponte et al., 2011, Arrigoni and Saper, 2014, Schone et al., 2014). Thus, we sought to gain insight into instantaneous spiking frequency in our sample of ARC neurons by considering the distribution of inter-spike intervals (ISIs). Most neurons (94/100) demonstrated tonic firing with unimodal ISI distributions (Figure 2��figure supplement 1; Hartigan's Dip Test, p